Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification - The authors discuss an alternative to traditional Protein A resins. - BioPharm International

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Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification
The authors discuss an alternative to traditional Protein A resins.


BioPharm International
Volume 24, Issue 5, pp. 36-42

POLYBIND–Z: AN UNUSUAL PROTEIN A RESIN AND ITS APPLICATION PERFORMANCE

For decades, the biologics industry has relied on packed-bed chromatography as the gold standard for antibody capture. Well-known, understood, and characterised materials and processes have enabled antibody manufacturers to manage the capture step of antibodies with predictable outcomes. The primary issue with traditional protein A sepharose media is that it is costly, requiring multiple cycles to reduce the overall cost contribution of the media to the final product. The need for these additional cycles has further complicated the process by requiring the introduction of wash, regeneration, sanitization, and validation steps to bring media costs to an acceptable level. Standard, packed-bed chromatography operates with pressures in the 0.1–0.4 MPa range, which is enabled because of the size, rigidity, and porosity of their solid supports. Typical commercial offerings are composed of beads that are usually >100 μm in diameter and that allow sufficient interstitial space for adequate flow rates. Balancing the high flow rate of larger particles and maintaining sufficient binding capacity is achieved by managing the porosity of the solid supports, effectively increasing surface area as well as increasing binding times due to diffusion of the target into the pores.

The functional beads can be delivered in a variety of formats in addition to bead slurries, based on initial experimental data One goal of our work has been to process the beads in such a way that their morphology is controlled, avoiding some of the limitations of the basic small particles, such as unsuitability for packed-bed columns or expanded-bed chromatography. In experiments, we processed the particles into fibres to be used as coatings, or cast into membranes. This work opens up a range of potential formats for the material for use alone or in combination with other materials, and processes. In addition to the use of the material for capture of antibodies, the same material could be used to remove impurities, or to catalyze reactions through the surface display of other binding proteins or enzymes, respectively.

We have developed simple batch purification protocols that allow the basic beads to be used in research or small-scale settings. Because of the surface display of the ligands, target binding is rapid and efficient, typically binding greater than 90% of the antibody in less than 30 seconds. This demonstrates the advantage of convective interaction over the current diffusive flow processes. Centrifugation at low speeds, <5000 x g, allows the beads to be resuspended and washed. A disadvantage in packed-bed chromatography, the beads' compressibility is an advantage when used in Eppendorf tubes because after binding, washing, and antibody release from the beads, they can be spun down (10,000 x g) to a point where they do not readily resuspend, allowing removal of the supernatant without aspiration of any beads.


Figure 3A: Static Immunoglobulin G (IgG) binding capacity (Eluted IgG in mg/g of drained beads). PB represents PolyBatics PolyBind–Z, 1 and 2 represent commercial protein A agarose beads. Binding capacity is in mg of human IgG/g of drained beads.
For commercial-scale application, we have seen early promise with the beads in a cross-flow filtration system. Initial experiments indicate this approach would be suitable for production- scale application of the particles in the capture of antibodies to produce a truly disposable harvest format.


Figure 3B: Commassie stained polyacryamide gel of unbound and elution fractions from a representative static hIgG binding experiment. 5 μg of purified huIgG (1 mg/ml) was incubated with 50 μg of either PolyBatics control beads (Lanes 3 and 4), Protein A sepharose beads (Lanes 6 and 7) or PolyBind–Z beads (Lanes 9 and 10) then eluted with equal volumes of a low pH buffer. Equal volumes of sample were loaded into each lane. Lane 1: Biolabs Ladder; Lane 2, 5 and 8: Pure IgG (5 μg); Lanes 3, 6 and 9 represent unbound fractions; Lanes 4, 7 and 10 represent elution fractions.
The engineered beads exhibit both high binding capacity and excellent specificity, with beads routinely exhibiting binding capacities in excess of 100 mg/g of drained beads (see Figure 3A). This level of binding represents a doubling of the published binding capacities of common commercial offerings, and is an artifact of the combined high surface-to-volume ratio and engineering of the fusion partners to exhibit more of the binding domains at the surface of the particles. We demonstrated the relative binding capacity of the PolyBind–Z versus two commercial offerings (see Figure 3A). In one example, we used 50 mg of drained PolyBind–Z to bind 5 mg of human IgG equating to a total binding of 100 mg/g drained PolyBind–Z beads (see Figure 3B).

In the case of the basic bead configuration, we displayed several of the Z–binding domains from protein A per engineered PS. It is also possible to combine specific binding domains, as one prototype combined the Z–binding domain of protein A with the GB1 binding domain of protein G to increase the range of IgG subclasses bound by the beads.

The specificity with which we have been able to remove IgG from the product feed stream is as important as the total binding capacity. In the case of human plasma, the PolyBind–Z was able to capture the IgG while leaving behind high abundance proteins, such as albumin.


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